Monitoring method and/or apparatus

ABSTRACT

A method and apparatus for substance monitoring. One application is an easy to handle continuous glucose monitor using a group of hollow out-of-plane silicon microneedles to sample substances in interstitial fluid from the epidermal skin layer. The glucose of the interstitial fluid permeates a dialysis membrane and reaches a sensor. Using MEMS technology, for example, allows well-established batch fabrication at low cost.

CROSS REFERENCE TO RELATED APPLICATIONS

This application a continuation of pending patent application Ser. No.12/188,918, which is a Divisional of U.S. Pat. No. 7,415,299 (A/N10/828,510) filed Apr. 19, 2004, which claims priority from provisionalpatent application 60/464,221 filed 18 Apr. 2003, each incorporatedherein by reference for all purposes. Priority is claimed to each of theabove applications.

The Invention was made with government support under Grant (Contract)No. F30602-00-2-0566 awarded by the Department of Defense. TheGovernment has certain rights to this invention.

COPYRIGHT NOTICE

Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of thisdisclosure contains material that is subject to copyright protection(such as, but not limited to, source code listings, screen shots, userinterfaces, or user instructions, or any other aspects of thissubmission for which copyright protection is or may be available in anyjurisdiction). The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or patent disclosure, asit appears in the Patent and Trademark Office patent file or records,but otherwise reserves all copyright rights whatsoever.

BACKGROUND OF THE INVENTION

The discussion of any work, publications, sales, or activity anywhere inthis submission, including in any documents submitted with thisapplication, shall not be taken as an admission that any such workconstitutes prior art. The discussion of any activity, work, orpublication herein is not an admission that such activity, work, orpublication existed or was known in any particular jurisdiction.

Currently proposed systems for monitoring substances of interest, suchas glucose, using small sampling and monitoring devices have a number ofdifficulties. For example, a microdialysis probe discussed for glucosemonitoring in U.S. Pat. No. 6,091,976, Jul. 18, 2000 (M. Pfeiffer and U.Hoss) is a needle-type probe with dialysis fluid flowing in and out ofthe probe. The probe is inserted at a length of several millimetersunderneath the skin at a shallow angle so that the probe stays in theepidermal tissue. A dialysis membrane separates the probe interior fromthe interstitial fluid surrounding the probe. This membrane allowsdiffusion of substances such as glucose from the interstitial fluid intothe dialysis fluid flowing in and out of the probe. The interstitialfluid is not extracted. The dialysis fluid is then pumped to a sensorplaced downstream where the glucose level of the dialysis fluid isdetermined. The glucose concentration of the dialysis fluid has beenfound to correlate with the glucose level in the interstitial fluid.

Despite the name microdialysis probe in this instance, the probedimensions are in the millimeter range. In these proposals, the reasonfor using such a long probe is that the area of the dialysis membranegenerally defines the amount of glucose diffusing into the dialysisfluid during a given amount of time. Generally, the detection limit ofpracticable glucose sensors requires a certain amount of glucose in thedialysis fluid to get reliable sensor signals. The required membranearea necessary for sufficient glucose diffusion and high sensor signalsis several square millimeters and this membrane generally defines thesize of the probe, which explains the large dimensions of the dialysisprobes and/or needles in these discussions.

A disadvantage of using a large “micro” dialysis probe is a generallypainful insertion procedure that generally requires trained personnel toimplant the probe underneath the skin. Thus, present microdialysisproposals do not easily allow for painless everyday usage.

According to the World Health Organization the per capita diabetes ratein the US increased from 5.2% (world: 2.4%) in 1995 to 6.0% (2.9%) in2000, and it is expected to reach 8.4% (4.5%) in 2030. While diabetes isthe leading cause of blindness, kidney failure and non-traumaticamputation of the lower limp, other severe complications associated withhyperglycemia (high glucose levels) and hypoglycemia (low glucoselevels) are nerve damage, heart disease, coma and brain damage. Thetraditional fingerstick test typically takes periodic samples, but thismonitoring can miss periods of hyperglycemia and hypoglycemia,especially during sleep. This health risk can be avoided using acontinuous glucose monitor.

Currently available continuous glucose monitoring systems include theCygnus GlucoWatch® and the Minimed CGMS™. However, it is believed thatthese systems cannot provide an accurate everyday glucose level controland still require periodic fingerstick tests for sensor recalibration.The GlucoWatch® is easy to use but it relies on reverse iontophoreticinterstitial fluid sampling through the skin, which is affected byfluctuating skin permeability as described in K. R. Pitzer, S. Desai, T.Dunn, S. Edelman, Y. Jayalakshmi, J. Kennedy, J. A. Tamada, R. O. Potts,Detection of Hypoglycemia with the GlucoWatch Biographer, Diabetes Care,Vol. 24, No. 5, 2001

The CGMS™ is generally not designed for daily usage; it requires trainedpersonnel to insert the sensor under the skin, as described in E.Cheyne, D. Kerr, Making ‘sense’ of diabetes: using a continuous glucosesensor in clinical practice, Diabetes Metab Res Rev, 18 (Suppl. 1),2002.

While frequent and long periods of hyperglycemic blood glucose levelscan account for many long-term complications, hypoglycemia can causesudden coma and brain damage. Periodic fingerstick tests often fail todetect all hypoglycemic and hyperglycemic events since glucose levelscan change rapidly. In particular, nocturnal hypoglycemia often remainsundetected.

SUMMARY

The present invention, in specific embodiments, involves novels methodsfor minimally invasive monitoring. In further embodiments, the inventionprovides a device and/or method for detecting and or monitoringsubstances of interest, particular substances in biological researchand/or clinical settings. In further embodiments, the invention providesa device and/or method using dialysis and out-of-plane microneedles toprovide an improved sensor.

In more specific embodiments, the invention involves a method and/orapparatus for monitoring of substances in interstitial fluid under theskin of a human or animal or under the outer layer of a plant usingout-of-plane microneedles. For humans and animals, this can allowpainless everyday usage.

In specific embodiments, the invention can be distinguished fromproposals describing generally a single microdialysis probe or needle.In the present invention, it is not necessary to insert a dialysis probeor needle underneath the skin. In specific embodiments of the invention,the dialysis portion of the device remains outside of the body, even ina very small monitoring system.

In other embodiments the invention relates generally to a method andapparatus for continuous monitoring of compounds in the epidermalinterstitial fluid. As a specific example, the invention relates to aminimally invasive method for sampling compounds from the epidermalinterstitial fluid using hollow out-of-plane microneedles and theapparatus for sampling and analyzing these compounds. A particularapplication of this invention is to continuously monitor the epidermalinterstitial fluid glucose level.

In further specific embodiments, the invention involves an array (usedherein to indicate any type of grouping) of out-of-plane microneedlesthat vertically penetrate a skin or other surface. In specificapplications, the microneedles are approximately 200 μm long, which, forexample, is sufficient to reach the epidermal interstitial fluid inhumans. In further embodiments, the invention involves microneedles thatare pre-filled with a liquid, such as a buffer solution, resulting in aliquid-liquid interface between the liquid inside the needle and theinterstitial fluid once the needle is inserted. Substances from theinterstitial fluid such as glucose can diffuse into the lumens of theout-of-plane microneedles. In further embodiments, a dialysis membraneis placed on an opposite side of a substrate from the microneedles.Thus, the membrane separates the needle lumens from the dialysis fluid,which is pumped past the membrane to the glucose sensor. The amount ofglucose diffusing through the out-of-plane microneedles, through themembrane and into the dialysis fluid is generally defined by the totalarea where diffusion can take place. This area is defined by the totalcross section of all needle lumens.

In further example embodiments, a group of microneedles in included in asystem along with a system and/or method for automatic calibration.Automatic calibration allows the system to provide reliable monitoringresults without the need for additional calibration methods, such as aneedle-stick test. According to specific embodiments of the invention,the dialysis system use in combination with the out-of-planemicroneedles facilitates sensor recalibration.

The present invention in specific embodiments provides a disposablesensor system that is minimally invasive and provides accurate sensorreadings and painless and easy sensor application. An example of such asystem system consists of hollow out-of-plane microneedles to sampleglucose from the interstitial fluid of the epidermis, an integrateddialysis membrane and an integrated electrochemical enzyme-basedflow-through glucose sensor.

In a further and very specific example embodiment, an array of betweenabout 600 to 1500 microneedles is placed on an approximately 8 mm×8 mmsubstrate. One advantage of using an array of out-of-plane microneedlesis that the resulting membrane area is large enough for effectivediffusion but the insertion of a number of out-of-plane microneedles ispainless since the needles are in fact very small, actually in themicro-meter range. In addition the needle array is easy to apply byfixing (e.g., by taping) or pressing the device onto the skin rather theinserting a dialysis probe at a shallow angle several millimeter longunderneath the skin. According to specific embodiments of the invention,a monitoring device using microneedles can be applied to the skin andeffectively sample substances in interstitial without penetrating deeplyenough to impact nerve endings.

While example detectors according to specific embodiments of the presentinvention are described herein as used for performing a biologicalassay, it will be understood to those of skill in the art that adetector according to specific embodiments of the present invention canbe used in a variety of applications for detecting substances ofinterests. These applications include, but are not limited to: detectingcontaminants in foodstuffs; detecting ripeness and/or the presence ofsugars in plants or plant parts; detecting the presence of a desiredsubstance (such as petroleum components) in an exploration operation;insuring the presence of desired elements in a manufacturing product,etc.

The invention and various specific aspects and embodiments will bebetter understood with reference to drawings and detailed descriptionsprovided in this submission. For purposes of clarity, this discussionrefers to devices, methods, and concepts in terms of specific examples.However, the invention and aspects thereof may have applications to avariety of types of devices and systems. It is therefore intended thatthe invention not be limited except as provided in the attached claimsand equivalents.

Furthermore, it is well known in the art that systems and methods suchas described herein can include a variety of different components anddifferent functions in a modular fashion. Different embodiments of theinvention can include different mixtures of elements and functions andmay group various functions as parts of various elements. For purposesof clarity, the invention is described in terms of systems that includedifferent innovative components and innovative combinations ofinnovative components and known components. No inference should be takento limit the invention to combinations containing all of the innovativecomponents listed in any illustrative embodiment in this specification.

In some of the drawings and detailed descriptions below, the presentinvention is described including various parameters of dimension and/orother parameters. These should be understood as illustrating specificand possible preferred embodiments, but are not intended to limit theinvention. Many devices and/or methods have variations in one or more ofthe detailed parameters described herein will be apparent to persons ofskill in the art having the benefit of the teachings provided herein andthese variations are included as part of the present invention.

All references, publications, patents, and patent applications citedand/or provided with this submission are hereby incorporated byreference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an example microneedle-based continuousmonitor wherein microneedle lumens are filled with interstitial fluid bycapillary action and a substance of interest diffuses through theintegrated dialysis membrane into dialysis fluid that is pumped past anintegrated enzyme-based flow-through sensor according to specificembodiments of the invention.

FIG. 2 is a schematic diagram of an example simplified microneedle-basedmonitor wherein a pre-filled system allows glucose diffusion through themicroneedles to an integrated two-electrode enzyme-based sensoraccording to specific embodiments of the invention.

FIG. 3 is a schematic diagram of an example microneedle-based continuousmonitor including a separate calibration fluid system according tospecific embodiments of the invention.

FIG. 4 illustrates an example schematic diagram of a sensor systemshowing three representative microneedles, a dialysis membrane, fluidreservoirs and pumps, according to specific embodiments of the presentinvention.

FIG. 5 illustrates an example microneedle component with a crosslinkedpolymer used as a dialysis membrane and an active membrane optionallywith immobilized enzymes according to specific embodiments of theinvention.

FIG. 6 illustrates an example sensor response to a fast decreasingglucose level (2.25 mg/di/min) showing that the time lag of the sensorresponse is approximately 2 min and thus, a hypoglycemia alarm could betriggered at 54.7 mg/dl according to specific embodiments of theinvention.

FIG. 7 illustrates the electrical operation of an example enzyme-basedelectrochemical glucose sensor that can be used in systems and/ordevices according to specific embodiments of the invention.

FIG. 8 illustrates an example of data showing sensor calibration (left)according to specific embodiments of the present invention.

FIG. 9 illustrates an example device with approximately 1000microneedles and other components according to specific embodiments ofthe present invention.

FIG. 10 is a scanning electron micrograph showing an example microneedleconfiguration of one configuration according to specific embodiments ofthe invention.

FIG. 11 is a scanning electron micrograph showing an example of analternative microneedles configuration (e.g., needles are approximately270 μm long, 100 μm wide shaft, ID=50 μm, 400 μm pitch) that can be usedin a dialysis system according to specific embodiments of the invention.

FIG. 12 is a schematic diagram of a skin penetration method using hollowout-of-plane microneedles according to specific embodiments of theinvention.

FIG. 13 is a schematic diagram of a skin penetration method using apre-bent elastic plate with through holes according to specificembodiments of the invention.

FIG. 14 illustrates aspects of a novel technique for in-device enzymeimmobilization which in this particular example is based on poly(vinylalcohol)-styrylpyridinium, a water-soluble photosensitive polymercontaining enzymes according to specific embodiments of the presentinvention. The figure can be understood to illustrate an sensor/dialysisportion of a sensor system to which a microneedle array may be attached.

FIG. 15A-B illustrates an example wafer stack with auxiliary channelsfor filling according to specific embodiments of the present invention.

FIG. 16 illustrates an immobilized heat-sensitive substance in sidepockets of a flow channel according to specific embodiments of thepresent invention.

FIG. 17 illustrates an immobilized heat-sensitive substance around pinsinside a flow channel according to specific embodiments of the presentinvention.

FIG. 18 is a block diagram showing a representative example logic devicein which various aspects of the present invention may be embodied.

FIG. 19 (Table 1) illustrates an example of diseases, conditions, orstatuses for which substances of interest can evaluated according tospecific embodiments of the present invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS 1. Definitions

The following definitions may be used to assist in understanding thissubmission. These terms, as well as terms as understood in the artshould be used as a guide in understanding descriptions provided herein.

A “substrate” is a, preferably solid, material suitable for theattachment of one or more molecules. Substrates can be formed ofmaterials including, but not limited to glass, plastic, silicon,germanium, minerals (e.g. quartz), semiconducting materials (e.g.silicon, germanium, etc.), ceramics, metals, etc.

2. Overview of the Invention

According to specific embodiments, the present invention involvesmethods, devices, and systems that enable a new approach to monitoringsubstances of interest from within an environment (such as a plant oranimal) by using out-of-plane microneedles and a sensing method that issubstantially external to the environment in which a substance is beingmonitored. A primary application is continuous glucose monitoring inhumans, though other applications are contemplated.

Integrated systems and/or methods of the invention generally comprise anarray of out-of-plane microneedles that are inserted into an area to besensed (such as skin), and integrated into the non-inserted side of themicroneedles one or more sensing components. The microneedles can be ofvarious configurations, examples of which are described herein. Thesensing components in their most simple state can include a prefilledbuffer reservoir with chemical and/or electrical sensor components.Other systems can include dialysis elements, electronic controls, smallscale or microfluidic channels, pumps, and systems, dialysis componentsand/or calibration components. A number of example configurations ofsuch integrated systems are described in detail below.

The invention is also involved with a number of novel techniques and/ordevices that enable or improve such monitoring systems in particularembodiments. These techniques and/or devices have applications and usesin different systems than the examples given here, as will be understoodby those of skill in the art from these teachings and in some cases areindependently novel.

3. Example System Configurations

To provide different contexts for understanding embodiments of thepresent invention, various example embodiment of sensing systems orportions thereof according to specific embodiments of the invention areillustrated in FIG. 1 through FIG. 5

In each case, these figures schematically represent the combination ofout-of-plane microneedle arrays with other components to form amicroneedle array detection system. Note that, in each of theseillustrations, the one to three microneedles illustrated should beunderstood to represent an array of generally tens, hundreds, or athousand or more microneedles as illustrated below. In some embodiments,a large set, up to all available microneedles, may be integrated with asingle detection system at the base of the needle. In other systems, twoor more separate detection systems can be integrated at the base of asingle microneedle array, either to provide different sensing, for easeof use or manufacturing, for staged use, or to provide a control system.

Dialysis

FIG. 1, FIG. 3, FIG. 4, and FIG. 5 each illustrate different embodimentsof a sensor system that includes a dialysis membrane to separate thesensing area from the sample area. This is a presently preferredembodiment. Dialysis is a well known technique for using a selectivelypermeable membrane between two fluids to allow diffusion of a desiredsubstance while preventing diffusion of other substances. One examplemembrane that can be used in systems according to specific embodimentsof the invention is an integrated porous polysilicon dialysis membrane,as will be understood in the art. Other example membrane technology willbe understood from the description herein and cited references. Insystems according to specific embodiments of the invention, the dialysismembrane is any membrane or system or structure that allows diffusion ofa substance that is intended to be detected and prevents one or morepossibly interfering substances.

Diffusion

FIG. 2 is a schematic diagram of an example simplified microneedle-basedmonitor wherein a pre-filled system allows glucose diffusion through themicroneedles to an integrated two-electrode enzyme-based sensoraccording to specific embodiments of the invention. While this systemmay not have the lifetime or reliability of the dialysis-based systemsin human applications, it has proven valuable as a prototyping designand has applications where ease of manufacturing and/or reduced cost areprimary considerations or where the sensor is used in applications thatdo not involve the presence of proteins or other compounds that cancontaminate the sensing components.

4. Operation Examples

As an example of operation, in these detectors, glucose or anothersubstance of interest that is present in blood or interstitial fluiddiffuses into the microneedles. This transport may be facilitated byprefilling the microneedles with a substance to aid diffusion (e.g., abuffer fluid or gel) to prevent air trapped in the needle lumen fromblocking fluidic flow or diffusion. With the lumen in contact with theinterstitial fluid, substances of interest can come in contact with oneor more sensors, such as chemical, electrical, electrochemical, optical,temperature, etc. sensors. In the example systems, example sensorsinclude the WE, CE and/or RE electrodes shown in FIG. 1, the integratedsensor components shown in FIG. 2. Catalysts or reagents can also beincluded depending on the type of sensing assay being used (e.g., theGOX regions shown in the figures).

Operation Example Details

A sensor system according to specific embodiments of the invention canhave a number of components depending on the particular type of sensorused. Systems including dialysis include a dialysis barrier and caninclude a dialysis fluid reservoir, fluidic channels, micropumps andvalues as shown. Systems including a calibration system can include acalibration fluid reservoir, fluidic channels, micropumps and values asshown. In some embodiments, calibration fluid is segregated from sampleor dialysis fluid by a moveable valve or by a flow restriction valve asshown. In alternative embodiments, calibration can be accomplished bychanging the flow rate of dialysis fluid and using that fluid forcalibration.

Example 1

FIG. 1 is a schematic diagram of an example microneedle-based continuousmonitor wherein microneedle lumens are filled with interstitial fluid bycapillary action and a substance of interest diffuses through theintegrated dialysis membrane into dialysis fluid that is pumped past anintegrated enzyme-based flow-through sensor according to specificembodiments of the invention. In this example, an array of hollowout-of-plane microneedles is used to penetrate the skin and to interfacewith the interstitial fluid. A dialysis membrane separates theinterstitial fluid and the dialysis fluid; thus, no interstitial fluidis extracted during operation. Dialysis fluid with a known constantglucose concentration is continuously pumped past the dialysis membraneand an integrated sensor (e.g., for glucose). Glucose diffuses throughthe microneedles and through the dialysis membrane into or out of thedialysis fluid. The concentration change in dialysis fluid ismeasured—it depends on the flow rate of the dialysis fluid and theglucose concentration in the interstitial fluid. At high flow rates(recalibrating mode) the amount of glucose diffusing into the dialysisfluid is negligible so that the glucose concentration of the dialysisfluid remains unchanged. Thus, a known concentration is measured and thesensor can be recalibrated. At low flow rates (measuring mode) theconcentration in the dialysis fluid changes significantly—the change inglucose concentration corresponds to the glucose concentration in theinterstitial fluid.

Example 2

FIG. 3 is a schematic diagram of an example microneedle-based continuousmonitor including a separate calibration fluid system according tospecific embodiments of the invention. In this further specific example,operation of the example sensor can be understood as follows. An groupof, for example, about 200 μm long hollow out-of-plane microneedles areused to penetrate the topmost layer of the skin, allowing their openingto come in contact with interstitial fluid from the epidermis. Once themicroneedles are either filled with interstitial fluid or oncesufficient time has elapsed for a substance of interest to diffuse intoprefilled needles, the substance of interest (e.g., glucose) diffusesthrough the dialysis membrane into dialysis fluid, keeping unwantedsubstances (e.g., larger protein molecules) outside of the dialysisarea, thus improving the sensor long-term stability.

Various detection strategies can be used for detecting substances ofinterest. Different strategies may be employed in different embodimentsof the invention. As one example, consider the enzyme-based flow-throughglucose sensor shown in FIG. 3. This sensor includes a Pt workingelectrode (WE), an Ag/AgCl reference electrode (RE) and a Pt counterelectrode (CE). The glucose oxidase (GOX) is immobilized upstream fromthe working electrode inside the flow channel.

In this example system, an integrated diffusion barrier channel preventsglucose diffusion from the calibration fluid into the dialysis fluidduring sensor recalibration. Other types of barriers, such as moveablevalves, etc., can be used in other embodiments, but a barrier as shownis easy to fabricate and effective in many situations. The diffusionbarrier consisting of a long and narrow diffusion path preventsdiffusion of glucose from the calibration fluid into the dialysis fluidduring sensor recalibration. In the figure, this barrier is shown withthe diffusion path oriented vertically in the dialysis fluid channel.While this provides an easy to view illustration, using typicalmicrofabrication techniques, the diffusion barrier will usually moreeasily be fabricated with the diffusion path oriented horizontally onthe substrate and thus the diffusion barrier path shown in FIG. 3 can beunderstood as a top down view of that portion of the system.

Electrical-Chemical Sensor

As an example of one type of sensor that can be used in a microneedlesystem according to specific embodiments of the invention, the sensorcomponents shown in FIG. 3 are further described. In the presence ofdissolved oxygen, glucose oxidase immobilized inside the channelcatalyses the oxidation of glucose to gluconic acid. Hydrogen peroxideis formed as a by-product.

Glucose+O₂

Gluconic acid+H₂O₂

The hydrogen peroxide is detected downstream using an integratedelectrochemical sensor. The working electrode is biased 0.7 V versus thereference electrode. Thus, hydrogen peroxide is oxidized at the workingelectrode and the resulting electric current is proportional to theglucose concentration inside the dialysis fluid.

H₂O₂

O₂+2H⁺+2e⁻

In this example system, an integrated diffusion barrier channel preventsglucose diffusion from the calibration fluid into the dialysis fluidduring sensor recalibration. Other types of barriers, such as moveablevalves, etc., can be used in other embodiments, but a barrier as shownis easy to fabricate and effective in many situations. The diffusionbarrier consisting of a long and narrow diffusion path preventsdiffusion of glucose from the calibration fluid into the dialysis fluidduring sensor recalibration. In the figure, this barrier is shown withthe diffusion path oriented vertically in the dialysis fluid channel.While this provides an easy to view illustration, using typicalmicrofabrication techniques, the diffusion barrier will usually moreeasily be fabricated with the diffusion path oriented horizontally onthe substrate and thus the diffusion barrier path shown in FIG. 3 can beunderstood as a top down view of that portion of the system.

Since the chlorine ion concentration in biological fluids remainsconstant at 0.15 mM a simple planar Ag/AgCl electrode can serve as apseudo reference electrode. For automatic sensor recalibration,reference glucose solution is periodically pumped past the sensor. Thus,no fingerstick tests are required to account for the usual gradual lossof enzyme activity during the sensor operation time.

Example 3

FIG. 4 illustrates an example schematic diagram of a sensor systemshowing three representative microneedles, a dialysis membrane, fluidreservoirs and pumps, according to specific embodiments of the presentinvention. In this example system, separate calibration and dialysisfluids reservoirs are used, with two micropumps and valves as shown.

Example 4 Microneedle with Cross-Linked Polymer

FIG. 5 illustrates an example microneedle component with a crosslinkedpolymer used as a dialysis membrane and an active membrane optionallywith immobilized enzymes according to specific embodiments of theinvention. In a particular example construction, the polymer iscrosslinked in the flow channel right underneath the needles where itforms walls around the needle lumen opening from the bottom to the topof this channel. In this configuration, the compounds from theinterstitial fluid diffuse through the needle lumen and through the gelwall where they might undergo enzymatic reactions before getting intothe dialysis fluid in the case where enzymes have been immobilized inthis membrane.

Thus, in this specific example, locally crosslinked polymer forms wallsin the flow channel underneath the needles, separating the interstitialfluid from the dialysis fluid. Analytes can diffuse through thispolymer.

Electrical Components

A prototype system using the glucose as describe above was tested. FIG.6 illustrates an example sensor response to a fast decreasing glucoselevel (2.25 mg/di/min) showing that the time lag of the sensor responseis approximately 2 min and thus, a hypoglycemia alarm could be triggeredat 54.7 mg/dl according to specific embodiments of the invention.

FIG. 7 illustrates the electrical operation of an example enzyme-basedelectrochemical glucose sensor that can be used in systems and/ordevices according to specific embodiments of the invention. Anotherexample glucose sensor is an integrated glucose sensor as discussed inM. Lambrechts and W. Sansen, Biosensors: Microelectrochemical devices,IOP Publishing, New York, 1992. Other sensor technology can be employedaccording to specific embodiments of the invention.

FIG. 8 illustrates an example of data showing sensor calibration (left)according to specific embodiments of the present invention.

In specific example systems, power supply and signal processing areachieved with a portable pager size device that connects to themicrosystem. The portable pager sized external device can also includecomponents for connecting to a computer and/or information processingsystem, either through a physical adaptor or wireless connection. Awireless connected device can be used in home and or office settings toallow an individual to be remotely monitored by, for example, a healthcare provider or elder care provider. A large number of such monitoringdevices can be used in institutional settings, such as care facilitiesand/or work environments and/or hospitals to monitor a number ofindividuals.

Micropumps

Techniques and/or devices for constructing micropumps are well-known inthe art and in general any micropumping technique can be included insystems according to specific embodiments of the invention. Examplemicropumps that can be used according to specific embodiments of theinvention are discussed in N.-T. Nguyen, X. Huang, T. K. Chuan,MEMS-Micropumps: A Review, Journal of fluids Engineering, Vol. 124,2002.

Valve

According to specific embodiments of the invention, a two-way valveconsisting of a diffusion barrier and a check valve allow pumping eitherdialysis fluid or calibration fluid to the sensor is employed. Thisvalve represents a novel design. Other valve designs can be incorporatedin specific embodiments of the invention. It should be understood that athe diffusion barrier as illustrated in FIG. 3 is schematically shownperpendicular to a substrate in order to illustrate its construction. Inspecific embodiments, this barrier will be constructed in a planeparallel to the largest substrate plane.

Integrated Systems

An example embodiment was fabricated using fabrication steps that willbe familiar in the art in addition to the teachings provided herein andin cited references. FIG. 9 illustrates an example device withapproximately 1000 microneedles and other components according tospecific embodiments of the present invention. Other processes,including processing having printing, molecular growth and/or otherfabrication steps as understood in the art can also be used to fabricatea device embodying the invention. Thus, FIG. 9 can also be understood asillustrating an early prototype of a simplified monitor, which onlyconsists of out-of-plane microneedles and a glucose sensor.

5. Microneedle Designs

A number of different methods are known for forming microneedles and avariety of these methods and different types of microneedle arrays canbe used in a device according to specific embodiments of the invention.One such device is described in B. Stoeber, D. Liepmann, Method ofForming Vertical, Hollow Needles within a Semiconductor Substrate, andNeedles Formed thereby, U.S. Pat. No. 6,406,638, Jun. 18, 2002.Microneedle arrays built using plastic and metal technology can also beused in a device according to specific embodiments of the invention.

FIG. 10 is a scanning electron micrograph showing an example microneedleconfiguration of one configuration according to specific embodiments ofthe invention. These microneedles can be used in specific embodiments ofthe invention and can fabricated as discussed in U.S. Pat. No.6,406,638.

It has been found is some situations, however, that longer and/orsharper microneedles may provide more easy penetration of varioussurfaces. FIG. 11 is a scanning electron micrograph showing an exampleof an alternative microneedles configuration (e.g., needles areapproximately 270 μm long, 100 μm wide shaft, ID=50 μm, 400 μm pitch)that can be used in a dialysis system according to specific embodimentsof the invention. These needles can be fabricated using the etchingtechniques disclosed in U.S. Pat. No. 6,406,638, but with etching stepsmodified to achieve longer and/or sharper needles. This new microneedledesign allows easier penetration of the skin due to a longer needleshaft, which causes the skin to stretch more and to break the stratumcorneum. In some cases, these longer microneedles may reach thecapillary bed of the dermis so that blood is sampled through the needlesalong with or instead of interstitial fluid.

While etched microneedle designs have been the most studied so far,other methods for forming microneedles can also be employed according tospecific embodiments of the invention.

In one such method, a liquid such as a polymeric fluid can be pouredonto a surface with thin pillars perpendicular to this surface.Different mechanisms can then be used to make this liquid higher aroundthe pins than further away from them to generate the needle shape andthe pins can then be removed after or during hardening of the liquid.The liquid could either be poured onto the molding surface from the top,it could enter from the side, or it could be pushed onto this surfacethrough bottom holes in this surface. It is also possible to condensateor to sublime this material on the molding surface. Capillary action cancause the liquid to rise up on the surface of the thin pillars, with theheight of rise will depend on the contact angle between the fluid andthe pillars, the surface tension of the fluid and its specific weight.The liquid can then be hardened in its current conformation.

6. Breaking Outer Surface or Membrane

In further embodiments, the invention involves a novel method forbreaking the outer layer of mammalian skin (stratum corneum) in order tocreate an interface with bodily fluids. This method consists of applyinga localized high pressure-load to one or multiple small location on theskin in order to yield the outer skin layer. This effect can be promotedby applying a preload to the skin in form of lateral stretching.

Large hypodermic needles are classic means for the penetration ofmammalian skin. This method has been used for injection as well asextraction of fluids from organisms. It requires sharp individualneedles, usually made from steel, which cut through the outer skin layerand open a passage for insertion of the needle shaft into the tissue.Some proposed microneedle methods replicate this method on a smallerscale, where needle shaft lengths were typically less that 1 mm. Thetarget depth in the tissue is typically not as deep as in the case ofhypodermic needles. It typically ranges from tens to only hundreds ofmicrons.

Effort has been spent on generating extremely sharp microneedles, whichcut the skin open in order to allow injection of fluids into theorganism or sampling of bodily fluids in the same fashion as in the caseof hypodermic needles. However, fabrication of extremely sharp smallneedles can be difficult and expensive. Furthermore, it is unclear ifthe sharp tips of these microneedles have a sufficient mechanicalstrength to prevent breakage during usage. In addition, the skin and theunderlying tissue are very flexible for small deflection as typicallycaused by short microneedles, so that the classical approach of cuttingthrough the stratum corneum risks to fail due to insufficient contactpressure. This problem is even more severe in the case of needle arrays,where a distributed load over a wide area of skin can results in a bedof nails effect, which merely leads to uniformly pushing down the skin.Nevertheless, microneedles allow easy integration into advanced drugdelivery systems or into systems for detection of body fluids and/orcompounds in an organism, which could be very important for the futureof medical care.

A number or alternative methods for skin penetration have beendeveloped, which use high-speed impact of some material onto the skin.The skin cannot deform rapidly because of its inertia and ruptures. A.B. Baker and J. E. Sanders (Fluid mechanics analysis of a spring-loadedjet injector, IEEE Transactions on Biomedical Engineering, 46 (2),February 1999, pp. 235-242) used the inertial force of a thin liquid jetto cut through the skin, M. A. F. Kendall, P. J. Wrighton Smith and B.J. Bellhouse (Transdermal ballistic delivery of micro-particles:Investigation into skin penetration, Proceedings of the 22^(nd) AnnualEMBS International Conference, July 23-28, Chicago, Ill., USA, pp.1621-1624, 2000) and X. L. Yu, X. W. Zhang, Y. Wang, J. Xie and P. F.Hao (Particle acceleration for delivery deoxyribonucleic acid vaccineinto skin in vivo, Review of Scientific Instruments, 72 (8), pp.3390-3395, 2001) drove small ballistic particles through the outer skinlayer into deeper tissue, and S. Lee, D. J. McAuliffe, T. Kodama and A.G. Doukas (In vivo transdermal delivery using a shock tube, Shock Waves,10, pp. 307-311, 2000) generated shock waves in order to enhance drugdiffusion into the skin. A more destructive method for opening the skinuses localized heat to burn a hole into the stratum corneum (M.Paranjape, J. Garra, S. Brida, T. Schneider, R. White, J. Currie,“Dermal thermo-poration with a PDMS-based patch for transdermalbiomolecular detection”, Technical Digest of the Solid-State Sensor,Actuator, and Microsystems Workshop 2002, Hilton Head Island, S.C., USA,June 2-6, pp. 73-76, 2002).

These results lead to the conclusion that breaking the stratum corneumwith shorter microneedles in order to provide diagnostic sensing may beimproved by using a different mechanism than simply penetrating thestratum corneum with needles. The needle tips are rather used togenerate high local stress in the stratum corneum without breaking it,while providing an additional load on the skin from a pressurized liquidinside the needles to rupture the skin.

This mechanism can be used for glucose sampling through the shortmicroneedles. In this approach, the outer skin layer can be broken byapplying high pressure to a small local skin region, which results inrupture of the cell matrix. This effect can be promoted by applying apreload to the skin in form of lateral stretching. Pressing such amicroneedle against the skin as shown in FIG. 12 (top) stretches theskin over the needle tip, so that additional pressure applied to thefluid inside the needle lumen results in yielding of the skin, whichruptures and opens a passage way between fluids inside the needle lumenand bodily fluids underneath the broken skin layer, FIG. 12 (middle).The stratum corneum slips back while the needle tip is inserted into theepidermis.

This opened passage can be used for multiple purposes. Compounds orfluids from within the organism can get transported through the needlelumen by diffusion or other transport mechanisms as shown in FIG. 12(bottom left), so that these compounds can be detected or quantified formonitoring purposes. Such compounds or fluids could be glucose, lactate,proteins, lipids, DNA, cells or blood.

This flow passage can also be used for injection of fluids into theorganism as shown in FIG. 12 (bottom right). In addition, this interfacewith bodily fluids can be used to send and/or collect electrical oroptical signals into or from the organism for detection purposes.Multiple needles in form of an array can be used simultaneously for anidentical purpose or multiple applications.

As a major advantage, this perforation method does not require extremelysharp microneedles, which allows simpler fabrication at low cost.Furthermore, less sharp microneedles are less susceptible to breakage oftheir tip increasing their reliability. In addition, the usage of lesssharp needles is safer since they only penetrate skin in response to thecombined forces of stretching the skin and pressurizing the fluid.

In certain cases it might be possible to apply this method of skinperforation without using microneedles. FIG. 13 shows an apparatus thatstretches the skin as it is being pressed against it. The base of thisapparatus extends laterally while its edges hold on to the skin. Thisbase also provides small trough holes, which can be used to applyadditional pressure to the small regions of the skin underneath theseholes by pressurizing a fluid from the side of the base opposite to theskin. Small rims around these openings on the side of the skin provide agood seal between the apparatus and the skin during pressureapplication.

7. Immobilization Technique

According to specific embodiments of the invention, wafer-levelfabrication of an integrated system of the invention is preferablyperformed using anodic bonding at relatively high temperatures, such asabove about 100° C. However, enzymes or substances of interest inintegrated systems according to specific embodiments of the inventionand in other BioMEMS and similar systems can be adversely affected atmaximum temperatures well below this temperature. Glucose oxidase, forexample, denatures at temperatures above 60° C.

Thus, in specific embodiments, the present invention involves a novelimmobilization technique that allows patterning inside microchannelsafter bonding or other high-temperature steps have been performed. Thistechnique is applicable in various applications, such as other BioMEMSthat require high temperature steps and the integration ofheat-sensitive bioactive materials.

FIG. 14 illustrates aspects of a novel technique for in-device enzymeimmobilization which in this particular example is based on poly(vinylalcohol)-styrylpyridinium, a water-soluble photosensitive polymercontaining enzymes according to specific embodiments of the presentinvention. According to specific embodiments of the invention, anin-device enzyme immobilization technique uses a photosensitivewater-soluble polymer, such as, for example, PVA-SbQ for example asdiscussed in K. Ichimura, A Convenient Photochemical Method toImmobilize Enzymes, Journal of Polymer Science: Polymer ChemistryEdition, John Wiley & Sons, Vol. 22, pp. 2817-2828, (1984). This polymeris generally is mixed with buffer solution (e.g., PBS, pH 7.4)containing a substance of interest to be immobilized, such as glucoseoxidase.

Basic example fabrication steps can be understood as follows: (1)High-temperature wafer bonding (e.g., Pyrex to silicon) and any otherhigh-temperature steps are performed; (2) Channels are filled withenzyme-polymer solution, and (3) crosslinking polymer under UV light orother energy source to form gels in which, optionally, enzymes areentrapped; (4) rinsing out unlinked solution.

More specifically, after high temperature processing and/or fabricationsteps (such as, anodic wafer bonding of a Pyrex cover and silicon base)areas and/or devices can be filled with this substance-polymer solution.In particular embodiments, auxiliary channels can be used connecting allregions of interest and thereby allowing filling of an entire wafer orlarge area thereof by capillary force through a single inlet in a shorttime and generally without bubble formation. FIG. 15A-B illustrates anexample wafer stack with auxiliary channels for filling according tospecific embodiments of the present invention.

In further embodiments, the polymer is selectively exposed to UV light(e.g., at 365 nm (600 mJ/cm²)) optionally through a transparent orpartially transparent material (e.g., a Pyrex cover) generally using ashadow mask to cover those areas where it is not desired to fix thesubstance. Thus, the substance of interest (e.g., an enzyme) isentrapped in locally formed gel regions. In a specific exampleembodiments, the low absorption of Pyrex at 365 nm makes an exposurethrough the glass cover possible. However a variety of differentmaterials can be used with different wavelengths of light depending onthe wavelength having the desired effect on the chosen polymer solution.Many combinations of transparency and useable polymer materials andlight wavelengths can be used in different embodiments of the inventionand the selection of a workable combination of these will be within theordinary skill of those in the art having benefit of this disclosure.

When desired during fabrication (e.g., after wafer dicing), the unlinkedenzyme-polymer solution can be rinsed out of the chips by soaking themin buffer solution for several hours.

Experiments using specific example embodiments show that the enzymeactivity remains constant, which means that only a negligible amount ofenzyme is washed out of the gel while soaking. Furthermore an exampletested gel does not swell and thus will not block the flow channelduring sensor operation when dialysis fluid flows through the sensor. Inparticular embodiments, maintenance of the gel in place is aided bycrosslinking it in side pockets or around pins inside the channel, anexample of which is shown in FIG. 16 and FIG. 17. This assists the gelto be held in place during operation. Other techniques to enhancegel-adhesion can be used, such as modifying the surface to which the gelshould adhere, either mechanically (e.g., by introducing roughness inthe surface, etc.) or chemically by changing the chemical properties ofthe gel or change the properties of the material and/or surface to whichthe gel should adhere.

In further embodiments, dicing the wafer stack into chips opens thein-plane fluid ports of each sensor device. Capillary tubes (0=360 μm)can be glued into the fluid ports. Furthermore, Side pockets or pins(anchors) inside the flow channels can be used to hold the gel in placewhen dialysis fluid (buffer) flows through the channels during sensoroperation.

In a specific example embodiment related to glucose detection aselsewhere described herein, in order to show that glucose oxidase issuitable for photochemical immobilization the effect of UV light (365nm) on the enzyme activity has been investigated. No significantdecrease in activity could be measured for exposure energies up to 18000mJ/cm². Thus glucose oxidase is very insensitive to UV light and theexposure energy of 600 mJ/cm² use to crosslink PVA-SbQ has no effect onthe enzyme activity. In addition the enzyme can preserve its activitysince it is only entrapped inside the gel and not crosslinked to it.

However, in specific embodiments, the effective activity of theimmobilized enzyme is reduced compared to enzyme in solution due to thediffusion limited glucose concentration inside the gel. For example, ithas been found that the effective activity of enzyme immobilized in a1.5 mm thick gel layer with a free surface of 40 mm² and 0.006 U glucoseoxidase drops by 70% compared to 0.006 U of free enzyme in buffersolution for the same pH value and the same glucose concentration of 90mM. Such activity loss can be compensated by a suitable sensor design,which guarantees a thin gel film with a large free surface area.Furthermore a high enzyme concentration is required in the gel to ensurea diffusion controlled amperometric current independent of the enzymeactivity. However, a large amount of enzyme results in an oxygen-limitedcurrent at higher glucose concentrations. For a thin film of 5 μl gelcontaining 0.025 U glucose oxidase the current approaches saturation ata glucose concentration of about 180 mg/dl

In further embodiments, the invention can be embodied in advancedenzyme-based BioMEMS, such as a continuous self-calibrating glucosemonitor. In specific embodiments, the enzyme is immobilized as discussedabove in a micro-scale flow channel. As will be understood from theabove, wafer-level fabrication of such BioMEMS with integrated fluidiccomponents can require bonding techniques at elevated temperatures suchas anodic bonding. This conflicts with the high sensitivity of enzymesto temperature. Glucose oxidase, for instance, starts to denature at atemperature of about 60° C. Thus, enzyme immobilization needs to beperformed after wafer bonding.

8. Diagnostic Uses

As described above, following identification and validation of a sensorfor a particular substance, including biological molecules such assugars, proteins, fats, or any substance of interest according to theinvention, in specific embodiments such detectors are used in clinicalor research settings, such as to predictively categorize subjects intodisease-relevant classes, to monitor subjects on a continuous basis todetect a substance of interest, etc. Detectors according to the methodsthe invention can be utilized for a variety of purposes by researchers,physicians, healthcare workers, hospitals, laboratories, patients,companies and other institutions. For example, the detectors can beapplied to: diagnose disease; assess severity of disease; predict futureoccurrence of disease; predict future complications of disease;determine disease prognosis; evaluate the patient's risk; assessresponse to current drug therapy; assess response to currentnon-pharmacologic therapy; determine the most appropriate medication ortreatment for the patient; and determine most appropriate additionaldiagnostic testing for the patient, among other clinically andepidemiologically relevant applications. Essentially any disease,condition, or status for which a substance or difference can be detectedin an interstitial fluid can be evaluated, e.g., diagnosed, monitored,etc. using the diagnostic methods of the invention, see, e.g. Table 1.

In addition to assessing health status at an individual level, themethods and diagnostic sensors of the present invention are suitable forevaluating subjects at a “population level,” e.g., for epidemiologicalstudies, or for population screening for a condition or disease.

Web Site Embodiment

The methods of this invention can be implemented in a localized ordistributed data environment. For example, in one embodiment featuring alocalized computing environment, a sensor according to specificembodiments of the present invention is configured in proximity to adetector, which is, in turn, linked to a computational device equippedwith user input and output features. In a distributed environment, themethods can be implemented on a single computer, a computer withmultiple processes or, alternatively, on multiple computers. Sensorsaccording to specific embodiments of the present invention can be placedonto wireless integrated circuit devices and such wireless devices canreturn data to a configured information processing system for receivingsuch devices. Such devices could, for example, be configured to beaffixed to a subject's body.

Kits

A detector according to specific embodiments of the present invention isoptionally provided to a user as a kit. Typically, a kit of theinvention contains one or more sensors constructed according to themethods described herein. Most often, the kit contains a diagnosticsensor packaged in a suitable container. The kit optionally furthercomprises an instruction set or user manual detailing preferred methodsof using the kit components for sensing a substance of interest.

When used according to the instructions, the kit enables the user toidentify disease or condition specific substances (such as sugars and/orfats and/or proteins and/or anti-gens) using patient tissues, including,but not limited to interstitial fluids. The kit can also allow the userto access a central database server that receives and providesinformation to the user. Additionally, or alternatively, the kit allowsthe user, e.g., a health care practitioner, clinical laboratory, orresearcher, to determine the probability that an individual belongs to aclinically relevant class of subjects (diagnostic or otherwise).

Embodiment in a Programmed Information Appliance

The invention may be embodied in whole or in part within the circuitryof an application specific integrated circuit (ASIC) or a programmablelogic device (PLD). In such a case, the invention may be embodied in acomputer understandable descriptor language, which may be used to createan ASIC, or PLD that operates as herein described.

Integrated Systems

Integrated systems for the collection and analysis of detection results,including detection or expression profiles, molecular signatures, aswell as for the compilation, storage and access of the databases of theinvention, typically include a digital computer with software includingan instruction set for sequence searching and/or analysis, and,optionally, one or more of high-throughput sample control software,image analysis software, data interpretation software, a robotic controlarmature for transferring solutions from a source to a destination (suchas a detection device) operably linked to the digital computer, an inputdevice (e.g., a computer keyboard) for entering subject data to thedigital computer, or to control analysis operations or high throughputsample transfer by the robotic control armature. Optionally, theintegrated system further comprises an electronic signal generator anddetection scanner for probing a microarray. The scanner can interfacewith analysis software to provide a measurement of the presence orintensity of the hybridized and/or bound suspected ligand.

Readily available computational hardware resources using standardoperating systems can be employed and modified according to theteachings provided herein, e.g., a PC (Intel x86 or Pentiumchip-compatible DOS,™ OS2,™ WINDOWS,™ LINUX, or Macintosh, Sun or PCswill suffice) for use in the integrated systems of the invention.Current art in software technology is adequate to allow implementationof the methods taught herein on a computer system. Thus, in specificembodiments, the present invention can comprise a set of logicinstructions (either software, or hardware encoded instructions) forperforming one or more of the methods as taught herein. For example,software for providing the described data and/or statistical analysiscan be constructed by one of skill using a standard programming languagesuch as Visual Basic, Fortran, Basic, Java, or the like. Such softwarecan also be constructed utilizing a variety of statistical programminglanguages, toolkits, or libraries.

FIG. 18 is a block diagram showing a representative example logic devicein which various aspects of the present invention may be embodied. FIG.18 shows an information appliance (or digital device) 700 that may beunderstood as a logical apparatus that can read instructions from media717 and/or network port 719, which can optionally be connected to server720 having fixed media 722. Apparatus 700 can thereafter use thoseinstructions to direct server or client logic, as understood in the art,to embody aspects of the invention. One type of logical apparatus thatmay embody the invention is a computer system as illustrated in 700,containing CPU 707, optional input devices 709 and 711, disk drives 715and optional monitor 705. Fixed media 717, or fixed media 722 over port719, may be used to program such a system and may represent a disk-typeoptical or magnetic media, magnetic tape, solid state dynamic or staticmemory, etc. In specific embodiments, the invention may be embodied inwhole or in part as software recorded on this fixed media. Communicationport 719 may also be used to initially receive instructions that areused to program such a system and may represent any type ofcommunication connection.

Various programming methods and algorithms, including genetic algorithmsand neural networks, can be used to perform aspects of the datacollection, correlation, and storage functions, as well as otherdesirable functions, as described herein. In addition, digital or analogsystems such as digital or analog computer systems can control a varietyof other functions such as the display and/or control of input andoutput files. Software for performing the electrical analysis methods ofthe invention are also included in the computer systems of theinvention.

Thus, a microneedle-based system according to specific embodiments ofthe invention can be employed as an effective glucose monitor using amicroneedle array and dialysis. Due to the optimum needle dimensions, itis sufficient to simply press the system onto the skin in order to reachthe desired location in the epidermis with an abundant amount ofinterstitial fluid. The nerve endings are located deeper in the skin sothat this procedure is painless. The glucose monitor can be attached toa skin location (for example, with a self-adhesive, medical tape, aband, etc.) by the patient himself without an assisted insertionprocedure.

Other Embodiments

Although the present invention has been described in terms of variousspecific embodiments, it is not intended that the invention be limitedto these embodiments. Modification within the spirit of the inventionwill be apparent to those skilled in the art. It is understood that theexamples and embodiments described herein are for illustrative purposesand that various modifications or changes in light thereof will besuggested by the teachings herein to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the claims.

All publications, patents, and patent applications cited herein or filedwith this submission, including any references filed as part of anInformation Disclosure Statement, are incorporated by reference in theirentirety.

1. A method of in vivo monitoring of an individual's interstitial fluidcomprising: creating a plurality of fluid paths through a stratumcorneum area of the individual's skin, the fluid paths each comprising adistal end in fluid communication with interstitial fluid of theindividual, a proximal end in analyte communication with a sensing arealocated outside of the patient's body, and an interior space extendingbetween the distal and proximal ends of the fluid path; allowing atleast one analyte to passively diffuse from the patient's interstitialfluid through the fluid paths and into the sensing area; and sensing aconcentration of the at least one analyte in the sensing fluid using asensor located at least partially in the sensing area.
 2. The method ofclaim 1 wherein fluid in the fluid paths is not moving when the at leastone analyte is passively diffusing there-through.
 3. The method of claim1 wherein there is no fluid movement between the distal ends of thefluid paths and the sensor when the at least one analyte is passivelydiffusing through the fluid paths and into the sensing area.
 4. Themethod of claim 1 wherein no interstitial fluid is extracted from theindividual during the diffusion and sensing steps.
 5. The method ofclaim 1 further comprising displaying analyte concentration informationremote from the stratum corneum area of the individual's skin.
 6. Themethod of claim 5 further comprising wirelessly communicating analyteconcentration information to a display.
 7. The method of claim 1 whereinthe sensing step is performed by a sensor in fluid communication withthe sensing area and the interior spaces of the fluid paths, the methodfurther comprising calibrating the sensor by moving sensing fluid intothe sensing area.
 8. The method of claim 7 further comprising movingsensing fluid out of the sensing area as sensing fluid is moved into thesensing area.
 9. The method of claim 1 further comprising attaching adevice comprising the sensor area and the sensor to the individual withadhesive.
 10. The method of claim 1 further comprising providing amembrane located between the distal ends of the fluid paths and thesensor.
 11. The method of claim 1 wherein creating the plurality offluid paths through the stratum corneum area of the individual's skincomprises applying a microneedle array to the stratum corneum area. 12.The method of claim 1 wherein the proximal ends of the fluid paths arein fluid communication with the sensing area.